The magnetic particle process is increasingly being used as a high-throughput process for the automatic isolation of nucleic acids, in which total nucleic acid (both DNA and RNA) is isolated from a biological sample matrix by reversible binding to SiOH-modified magnetic particles. For this purpose the nucleic acids to be isolated are contacted with silica-modified magnetic particles in a chaotropic binding buffer. The binding of the nucleic acids to the particle surface takes place over a range of temperatures, from ˜18° to ˜38° C. for example, over a period of time up to an hour while the particle suspension is mixed by shaking or vortexing. The particles loaded with nucleic acids are then drawn towards the vessel wall by applying a magnetic field, and the supernatant is aspirated and discarded. After removing the magnetic field, the particles are resuspended and washed several times with a washing buffer or buffers. The nucleic acids bound to the magnetic particles are then removed from the particles at a high temperature, such as for example at 90° C. for 10 mins, with the aid of an elution buffer. After re-applying the magnetic field, the eluate containing the nucleic acids can be pipetted off. This process is described in detail in WO 2003/058649.
Ideally, magnetic particles for the automated isolation of nucleic acids (NA) are distinguished by a balanced combination of specific requirements with regard to particle size, silica content on the particle surface, magnetic properties, and purity. Magnetic particles of Fe3O4 (magnetite), such as for example the Bayoxide E types from Lanxess, which are used as electrographic toners, are commercially available magnetic particles that fulfill these properties to a certain degree.
The primary particle sizes of the Bayoxide E magnetic particles as determined by electron microscopy are in the range from about 0.2 to about 0.4 μm. This corresponds to a (BET) specific surface area in the range of a few m2/g, i.e. of about 4 to 12 m2/g, with the particularly preferred Bayoxide E 8706 particles having a specific surface area of 7 to 9 m2/g. Useful suspension stabilities can be obtained with magnetic particles having such particle size distributions.
On the one hand, in order to obtain the maximum possible quantitative binding of nucleic acids, the suspension stability should be such that the suspension of particles obtained by shaking in the binding buffer is as homogeneous as possible. On the other hand, the magnetic particles loaded with nucleic acids have to be completely removed within a short time after applying the magnetic field, such as within one minute, in order to obtain the shortest possible analysis time for high throughput methods.
Although magnetic particles with smaller diameters (e.g., “magnetic nanoparticles” or “nanoparticulate Bayoxides”) form highly stable suspensions, they require considerably longer times for their removal by the magnetic field. Magnetic particles with larger particle sizes (e.g., several μm) form suspensions whose stability is too low, which can have a negative effect on the adsorption of the nucleic acids.
Although the abovementioned Bayoxide E magnetic particles (e.g., Bayoxide E 8706 and Bayoxide E 8707) are suitable for nucleic acid analysis, these products also contain small quantities of nanoparticulate components which manifest themselves in the form of black dust. These nanoparticulates can bind considerable quantities of DNA due to their large surface area, yet they can only be removed with great difficulty by the magnetic field. Considerable losses in yield are therefore likely to occur in the nucleic acid isolation process as a result of these nanoparticulate components.
Commercially obtainable Bayoxide E magnetic particles also have been found to contain an additional form of nanoparticulate impurity, namely yellow-colored boehmite crystals (α-FeOOH). Boehmite crystals can be obtained in varying quantities depending on the production batch, and are formed due to a side reaction during the synthesis of magnetite (i.e., by the oxidation of FeSO4 at alkaline pH values). These particles are not magnetic but can bind nucleic acids, particularly when they are treated with silica, and therefore prevent a certain fraction of the nucleic acids in a sample from being removed magnetically.
Residues of the starting product, iron sulfate, have also been found in Bayoxide E magnetic particles as a third type of impurity. Although these iron salts cannot bind nucleic acids they are nevertheless disadvantageous since they can poison the enzymes used in PCR, which is frequently used for subsequent detection. Also, iron ions can form colored secondary products with the chaotropic buffer systems frequently used for nucleic acid isolation, which may contain guanidinium isothiocyanate, for example. These secondary products can considerably interfere with photometric analysis used in nucleic acid detection processes.
Aqueous suspensions of the abovementioned Bayoxide E particles are also disadvantageous because they have a relatively high affinity for vessel walls of glass or plastic, and considerable quantities of the magnetic particles can be adsorbed to such walls. More advantageous particle suspensions are those which flow off vessel walls leaving as little residue as possible, especially from microtiter plates made of thermoplastics, which are frequently used for nucleic acid isolation.
Industrially produced magnetic particles also exist which, as a result of the method employed for their production, contain small quantities of silica and display a certain nucleic acid binding capacity. In the synthesis of Bayoxide E 8706 and Bayoxide E 8707, for example, waterglass (an alkali metal silicate solution) is added in order to render the particles more spherical and less sharp-edged. The silica concentration available on the surface, and thus the nucleic-acid-binding capacity, of these particles is low, however, and also varies from batch to batch. Due to the resulting reduced nucleic acid binding capacities, relatively large quantities of such magnetic particles would have to be used for nucleic acid isolation, making it difficult or impossible to effectively process smaller volumes of nucleic acid samples.
Thus, there remains a need for magnetic particles with appropriate nucleic acid binding properties, magnetic separation properties, and freedom from chemical contaminants in order to improve yield, consistency, and throughput during nucleic acid isolation and analysis.